This invention relates to new vaccines against microorganisms based on antigenically mimetic peptides. The invention also relates to methods of discovering such mimetic peptides by screening peptide-display phage libraries with antibodies against the microbial carbohydrates(s) of interest to locate antigenically mimetic peptides. Vaccines against Group B Streptococcus, or Streptococcus Agalactiae, can be produced using this method. Vaccines against other microbial pathogens may also be produced using this method.
Vaccines protect against disease by harnessing the body""s innate ability to protect itself against foreign invading agents. During vaccination, the patient is injected with antigens, or DNA encoding antigens, which generate protective antibodies but which typically cannot cause severe disease themselves. For example, vaccination for bacterial diseases such as typhoid fever consists of injecting a patient with the bacterial agents of these diseases, after they have been disabled in some fashion to prevent them from causing disease. The patient""s body recognizes these bacteria nonetheless and generates an antibody response against them.
It is not always possible, however, to stimulate antibody formation merely by injecting the foreign agent which causes the disease. The foreign agent must be immunogenic, that is, it must be able to induce an immune response. Certain agents such as tetanus toxoid are innately immunogenic, and may be administered in vaccines. Other clinically important agents are not immunogenic, however, and must be converted into immunogenic molecules before they can induce an immune response. Successfully accomplishing this conversion for a variety of antigens is a major goal of a great deal of immunologic research.
However, researchers have yet to successfully convert a variety of poorly immunogenic antigens into optimally immunogenic molecules. Of particular importance to the present invention is the failure of immunologic researchers to successfully convert carbohydrates into optimally immunogenic molecules.
Carbohydrates are poorly immunogenic largely because of the way in which they interact with the body""s immune system. Carbohydrates frequently function as T-independent antigens, which cannot be properly processed by the antigen presenting cells that begin the typical mammalian immune response. By contrast, T-dependent antigens are initially processed by antigen presenting cells and then rely on T-cells to stimulate B cells to manufacture large quantities of antibodies against the antigen. As a result of these molecular biological differences, T-dependent antigens are immunologically superior to T-independent antigens, including carbohydrates, in three ways:
(1) T-dependent antigens are remembered by the immune system while T-independent antigens are not. Thus, after vaccination, an infection with a T-dependent antigen will be met with an extremely swift and concentrated antibody attack compared to the response to the initial vaccination. Infections with T-independent antigens, by contrast, generally receive the same level of antibody response, even after vaccination;
(2) T-dependent antigens are met with specific antibodies of increasing affinity against them over time, while T-independent antigens are met with antibodies of constant affinity; and
(3) T-dependent antigens stimulate a neonatal or immature immune system more effectively than T-independent antigens.
One approach which researchers have taken to enhance the immune response to T-independent antigens is to inject subjects with polysaccharide or oligosaccharide antigens that have been conjugated to a single T-dependent antigen such as tetanus or diphtheria toxoid. (Kasper, D., et al., J. Clin. Invest., Vol. 98, No. 10 2308-2314, 1996) (Schneerson, R. et al., Inf. Immun. 52:519, 1986) (Anderson, P W, et al., J. Immunol. 142:2464, 1989). These conjugate vaccines improve on vaccines based on carbohydrates alone because they xe2x80x9ctrickxe2x80x9d the T-cells into directing the immune response, giving this response something of the character of a T-dependent response, even though it is directed against a T-dependent/T-independent conjugate. However, this xe2x80x9ctrickxe2x80x9d is imperfectxe2x80x94although T-cells do assist, their assistance against conjugates is not as effective as it is against true T-dependent antigens. As a result, generally only low levels of antibody titres are elicited, and only some subjects respond to initial immunizations. Thus, several immunizations are frequently required. This poses a serious obstacle because patients are not always willing, or able, to complete this entire process; this is often true, for example, of patients who live a great distance from medical facilities, as is frequently the case for patients in lesser developed nations. And even when patients do complete the process, there is no guarantee of successxe2x80x94infants less than two months of age may mount little or no antibody response even after repeated immunization. Furthermore, the process itself sometimes takes so long that patients contract the disease in a virulent form before they have been properly vaccinated.
In another attempt to gain the advantages of T-dependent response with T-independent antigens, including carbohydrates, researchers have attempted to discover T-dependent antigens which are structurally related to the T-independent antigen of interest. In theory, these structural mimics might elicit a superior immune response, compared to a vaccine based on either the original T-independent antigen alone or as part of a conjugate. Under this approach, at least, no part of the antigen in the vaccine is incompatible with T-cell assistance.
Yet locating T-dependent antigens which are sufficiently structurally related to T-independent antigens to be true immunological mimics has proven difficult. Researchers have taken three different approaches to this problem, each of which has serious limitations.
First, some researchers have succeeded in designing synthetic peptides which are immunologically mimetic by using computer simulations and protein databases to construct a protein structure which closely resembles the structure of the T-independent antigen of interest, as ascertained through x-ray crystallography. (Westeruik et al., Proc. Nat. Acad. Sci. USA Vol. 92, 4021-4025, 1995). However, this approach is only as good as the researcher""s knowledge of the various structures involved, which is frequently far from complete. Furthermore, because even a single amino acid error can have a profound effect on the immunogenicity of the synthetic peptide, as Westernik notes, a very high level of precision is requiredxe2x80x94higher than may be possible for molecular systems whose structure is not well understood.
Second, some researchers have generated immunologic mimics by isolating anti-idiotypic antibodies which can elicit an immune response to carbohydrate antigens of S. pneumonia (McNamara et al., Science 226:1325, 1984), P. aeruginosa (Schreiber et al, J. Inf. Dis. 164:507, 1991), E. coli (Kacack, M. B. et al., Infec. Immun. 61:2289, 1991) and Group A Streptococci (Manafo, W. J. et al., J. Immunol. 139:2702, 1987). Anti-idiotypic antibodies are known to be structurally similar to the antigens of interest because of their design: they are generated against the idiotypes of antibodies which are known to specifically bind the carbohydrate of interest. As a result, the anti-idiotypic antibody and the carbohydrate bind specifically to the same idiotype structure (an antigenic determining structure in the antigen-binding portion of the carbohydrate binding antibody). Thus, much as two keys which fit the same lock have a high level of structural similarity, anti-idiotypic antibodies are thought to be structurally similar to the antibody-binding structures on carbohydrates. However, the similarity is not complete: these are still antibodies, isolated from the cells of mice, not complete carbohydrate structural mimics. As a result, there has been some concern that, for treatment of humans, human vaccines based on anti-idiotypic antibodies would be undesirable because of serious allergic reactions which could result. (Westernik, M. A. et al., Proc. Nat. Acad. Sci. USA vol. 92, 4021, 1995.) This concern has led at least some researchers to seek alternative means of discovering T-dependent antigens which are structurally similar to T-independent antigens. (Westernik, M. A. et al., Proc. Nat. Acad. Sci. USA vol. 92,4021, 1995).
Finally, some researchers have sought to discover T-dependent antigens which are structurally similar to T-independent antigens by screening libraries of phages, which express hundreds of millions of random peptide sequences, using known carbohydrate-binding antibodies to find particularly promising peptides. (Harris, S. et al., Proc. Natl. Acad. Sci. vol. 94, no. 6 pp. 2454-2459, 1997) (Valuation, P. et al., J. Mol. Biol. 261: 11-22, 1996) (Hoess, R. et al, Gene 128:43, 1993). (See Oldenberg, K. R., Proc. Nat. Acad. Sci. USA 89:5393, 1992 (using lectins to screen such libraries)). The approach outlined in these references is sound only if one accepts that antigenic mimicry (meaning that the peptide mimic binds the same highly specific antibody as the carbohydrate of interest) is reasonably predictive of immunologic mimicry (meaning that the peptide will generate an immune response against the carbohydrate of interest). After all, if antigenic mimics are only rarely immunologic mimics, this procedure leaves one with far more peptide sequence candidates for immunologic testing after the antigenic screening step than can reasonably be tested. Indeed, after several failed attempts at obtaining an immunologic mimic using this approach were conducted, many in the art have in essence concluded that this approach is fundamentally flawed. In particular, at least one researcher has concluded that antigenic mimicry is rarely predictive of true immunologic mimicry, because the mechanism of peptide-antibody binding is different than carbohydrate-antibody binding. (See Harris, S. et al., Proc. Natl. Acad. Sci. Vol. 94 No. 6 pp. 2454-2459, 1997).
Another serious limitation of both this approach and the design-approach of Westernik is that there is no a priori reason to believe that a peptide-based structural mimic necessarily exists for any given carbohydrate. The molecular basis underlying mimicry is unknown, and as such, offers no assurance that all carbohydrates structures have peptide mimics. There is certainly evidence in nature that some carbohydrate structures possess protein mimics. For example, the protein tendamistat is known to bind to the enzyme xcex1-amylase at the same location this enzyme binds carbohydrates. And further research with synthetic peptides has demonstrated a certain level of mimicry in a variety of carbohydrates drawn from a number of species, although the theoretic basis for much of this data has been questioned. (See Harris, S. et al., Proc. Natl. Acad. Sci. vol. 94, no. 6 pp. 2454-2459, 1997). Nevertheless, from these studies, it appears that each new carbohydrate presents a unique challenge to this area of research.
Partly as a result of all of these limitations, there remains a need in the art for vaccines effective against T-independent antigens and a method for developing such vaccines.
This need is particularly acute for vaccines effective against Group B Streptococci (GBS). Efforts at making a vaccine against GBS have focused on using the T-independent polysaccharide of GBS. However, as is frequently the case with T-independent antigens, vaccines containing only GBS polysaccharides have been only marginally effective in inducing antibody. (Baker, C. J. et al., New Eng. J. Med. 319:1180). Conjugate vaccines containing the GBS polysaccharide conjugated to tetanus toxoid, a protein carrier, have been more successful. (Kasper, D. L. et al., J. Clin. Invest. 98:2308). Nevertheless, there is considerable room for improvement in this area of the art.
This unmet need for novel vaccines against Group B Streptococci, or Streptococcus agalactiae, is only compounded by the widespread and frequently deadly infections attributed to this bacterial agent. The Center for Disease Control has recently declared prevention of GBS infections a major public health priority. (CDC, Morbidity and Mortality Weekly Report 45 (No. RR-7):1, 1996.) GBS causes invasive infections of newborns, pregnant women, and adults with underlying medical conditions. Although the bacteria are sensitive to antibiotics such as penicillin, case fatality rates are estimated to be 5-20% in newborn children and 15-32% in adults. Infection is most commonly seen as bacteremia, meningitis, and pneumonia. Newborns who survive the disease may suffer permanent neurologic sequelae as a result of meningitis. When mothers lack protective anti-GBS antibodies, their newborn children are at risk of infection. (Baker, C. J. et al, New Eng. J. Med. 294:753, 1976) (Baker, C. J. et al., J. Infect. Dis. 136:598, 1977) (Hemming, V. G. et al., J. Clin. Invest. 58:1379, 1976). The development of maternal vaccines is considered a leading approach to the prevention of GBS disease in newborns. (Mohle-Boetani, J. C. et al., J. Am. Med. Assoc. 270:1442, 1993.) Passive administration of antibody has defined protective epitopes of GBS (Pincus, S. H. et al., J. Immunol. 140:2779, 1988) (Shigeoka, A. O. et al., J. Infect. Dis. 149:363, 1984) (Shigeoka, A. O. et al., Antibiot. Chemother. 35:254, 1985) (Egan, M. L. et al., J. Exp. Med. 158:1006, 1983) (Raff, H. V. et al., J. Exp. Med. 168:905, 1988) (Lancefield, R. C. et al., J. Exp. Med. 142: 165, 1975), responses to which are critical for an effective vaccine.
Thus, there remains in the art a need for improved vaccines against GBS and methods for producing them.
The present invention addresses this unmet need for novel vaccines by providing a method to isolate a peptide which immunologically mimics GBS. This is the first demonstration that a peptide structural mimic exists for this bacteria.
In addition, this invention meets a more general need in the art because it succeeds in locating mimics where the other two general methods for isolating immunological mimics would fail, or result in suboptimal mimics. First, this invention can succeed in locating an immunologic mimic even when the structure of the carbohydrate antigen is unknown or when an immunologic mimic of that structure cannot be constructed from protein databases and computer simulations, unlike the method of Westernik et al. Second, this invention ultimately results in a vaccine which does not have the same potential for serious allergic reaction possessed by anti-idiotypic antibody based vaccines.
The present invention also offers a validation of the final method to isolating mimetic proteins by contradicting the findings of Harris, et al. Harris concludes that the binding of peptides is by a different mechanism than binding of carbohydrate, and that this is neither antigenic nor immunologic mimicry. (Harris, S. et al., Proc. Natl. Acad. Sci. Vol. 94 No. 6 pp. 2454-2459, 1997.) Harris et al. bases this conclusion on observations made when isolating peptides mimicking group A streptococcal cell-wall polysaccharide. Harris et al. observes that most peptides isolated as potential antigenic mimics were primarily reactive with the antibody used to isolated it, but only weakly reactive with other antibodies against the same cell-wall polysaccharide.
However, the data set forth below contradicts this data, as well as the conclusions drawn from it. In particular, it shows that two out of three IgG monoclonal antibodies to type III GBS also bind to the peptide isolated by the method of the present invention. (FIG. 1b). This data indicates that, while each of the monoclonal antibodies binds to the same polysaccharide structure, some recognize different aspects of that structure. This interpretation was considered by Harris, et al. but discarded in favor of Harris""s conclusion that the binding of peptides is by a different mechanism than binding of carbohydrate. (Harris, S. et al., Proc. Natl. Acad. Sci. Vol. 94 No. 6, at 2459, 1997.) Moreover, the data set forth below demonstrates that polyclonal anti-GBS antibodies bind well to the peptide mimetic. Thus the present invention contradicts the Harris findings.
The present invention relates to a method of isolating a peptide which immunologically mimics a portion of Group B streptococci, comprising the steps of:
(1) identifying protective antibodies reactive with said Group B streptococci;
(2) contacting a phage-display library, having phage, with one or more of said protective antibodies identified in step 1;
(3) isolating one or more phage, having a displayed peptide, which bind one or more of the protective antibodies; and
(4) selecting, for all said phage isolated in step 3, the peptides or peptide fragments to which the antibodies have bound.
Thus, the invention includes all mimetic peptides and peptide fragments that induce antibodies to GBS. Although any suitable portion of the GBS, such as lipotechoic acid or proteins, may be employed in the invention, in a preferred embodiment, the mimetic peptides induce antibodies against the GBS carbohydrate. In another embodiment, the mimetic peptide induces antibodies to the type III polysaccharide of GBS. In a more preferred embodiment, the invention relates to peptides having the sequence FDTGAFDPDWPA (SEQ ID NO: 1) or FDTGAFDPDWPAC (SEQ ID NO:2) or WENWMMGNA (SEQ ID NO:3) or WENWMMGNAC (SEQ ID NO:4) and fragments and derivatives thereof which exhibit the same or similar ability.
The present invention also relates to a vaccine consisting of these peptides and/or peptide fragments and/or derivatives together with a pharmaceutically acceptable carrier.
In yet another embodiment, the peptides and/or peptide fragments and/or derivatives of the invention may be conjugated to a carrier. In a further embodiment, multiple copies of the peptide are used. In another embodiment, a fusion protein containing the peptide is employed. In yet another embodiment, the vaccine uses DNA encoding for the peptide, the conjugate, or the fusion protein.
The present invention also relates method of treating a patient, comprising administering to the patient an immunostimulatory amount of the vaccine of the invention.
FIG. 1 Binding of antibodies to synthetic peptide FDTGAFDPDWPAC (SEQ ID NO:2).
Microtiter wells were coated with peptide at 10 xcexcg/ml and then blocked with 1% BSA. Test antibodies were added to the wells, incubated and washed. Antibody binding was detected with alkaline phosphatase conjugated anti-Ig and then substrate. The values are A405, mean of duplicate or triplicate samples. The binding of monoclonal antibodies S7 and S9 is shown in panel A. Panel B shows the binding of three IgG anti-GBS type III and one irrelevant monoclonal antibodies, and panel C shows the serum from mice infected with 108 live GBS type III (primary, secondary, and tertiary refer to the number of times the mice were infected). The binding of all antibodies to BSA was less than 0.1.
FIG. 2 Competitive inhibition assays. Panel A shows inhibition of S9 binding to GBS by peptide. Antibodies S7 or S9 were diluted to the concentrations shown and mixed with the indicated concentration of peptide FDTGAFDPDWPAC (SEQ ID NO:2). Following a one hour incubation, the peptide and antibody were transferred to GBS-coated microtiter wells, and incubated overnight. The plates were washed and antibody binding was detected with alkaline phosphatase-conjugated anti-mouse IgM and substrate. The values are A405, mean of duplicate samples. Panel B shows the inhibition of anti-GBS antiserum (secondary bleed from panel 1 C) binding to FDTGAFDPDWPAC (SEQ ID NO:2). The indicated dilutions of serum were mixed with intact GBS or purified type III capsular polysaccharide (III-CPS) and plated into peptide-coated wells. The plates were washed and antibody binding was detected with alkaline phosphatase anti-mouse Ig and substrate. The values are the mean and SEM of triplicate samples (if no error bars are seen, the SEM is too small to be drawn).
FIG. 3 Immunization of mice with peptide-carrier conjugates results in the production of GBS antibodies. Mice were immunized with peptide FDTGAFDPDWPAC (SEQ ID NO:2) conjugated to OVA (mouse 1 and 2), KLH (mouse 3 and 4), or BSA (mouse 5 and 6), or with live GBS, once (1xc2x0) or twice (2xc2x0). Both prebleed and post-immune sera were diluted 1:1000 and tested for binding to type III GBS (left panel) or to type III capsular polysaccharide (right panel).